Page 219 - Advances in Textile Biotechnology

200 Advances in textile biotechnology

non-aggregating, non-immunogenic, and more stable to proteolytic degra-
dation (Fontana et al., 2008; Mero et al., 2009). Interestingly, modifi cation

or PEGylation of protein-bound Gln residues occurred at specific sites. For
example, it has been shown that in apomyoglobin only Gln 91 was modifi ed
and in human growth hormone only Gln 40 and Gln 141, despite these
proteins having many more Gln residues. These studies shed light on the
molecular features dictating the selective attack of protein-bound Gln res-
idues by TGases and highlighted the close molecular similarities between
TGase-mediated reactions and proteolysis. In fact, TGase can attack the
same sites/regions of the polypeptide chain that suffer proteolysis, provided
that a Gln residue is encompassed by that chain region. Enhanced chain

flexibility, extended chain conformation, local unfolding/disorder of the
protein secondary structure such as in connecting loops between hydrogen-
bonded structures (helices) are the key parameters driving the TGase
attack to Gln residues. Thus, surface exposure alone does not seem a suf-

ficient condition for TGase to use a Gln residue as substrate, as shown by
the fact that not all surface exposed Gln residues in globular proteins are

modified by the enzyme. Only those surface-exposed residues that are

encompassed in a flexible or unfolded region can be attacked by TGase.

These findings entail important consequences because it appears possible
not only to explain but also to predict the sites of TGase-mediated modifi -
cation of globular proteins of known sequence and structure, thus opening
the prospect to properly design useful strategies for their modifi cation.

Biofabrication
The integration of proteins with microfabricated devices is an attractive
biofabrication approach for developing a range of applications, from pro-

teomics to microfluidics, biosensors, medical diagnostics, microarrays,
bioMEMS (biological microelectromechanical systems), and metabolic
engineering. An elegant example of the enzyme-mediated controlled assem-
bly of proteins is the stepwise assembly at electrode addresses of biological
polymers, such as chitosan and proteins, by a combination of electrical
stimuli and enzymatic reactions catalysed by tyrosinase and TGase (Yang
et al., 2009). Chitosan is considered a good interface for assembling proteins
at solid surfaces. The formation of a thin and resistant chitosan fi lm onto
solid electrode surfaces can be simply achieved by electrodeposition. Fur-
thermore, proteins can be assembled onto the polysaccharide layer by com-

bined enzymatic reactions that first exploit the nucleophilicity of the
chitosan primary amine to bind a peptide tether via the tyrosinase-catalysed
reaction, followed by the formation of a covalent bond between the tether
and another protein via microbial TGase-catalysed conjugation. The latter
protein could be an enzyme, a receptor, an antigen or an antibody, which

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